ABSTRACT: Here we determine the map of RNA methylation (m6A) in mouse embrionic stem cells, and Mettl3 knock out cells Examination of m6A modification sites on the transcriptome of mouse Embryonic stem cells and Embryonic Mettl3 knock out cells, using a m6A specific antibody.
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNAs. Despite its functional importance in various physiological events, the role of m6A in chemical carcinogenesis remains largely unknown. Here we profiled the dynamic m6A mRNA modification during cellular transformation induced by chemical carcinogens and identified a subset of cell transformation-related, concordantly modulated m6A sites. Notably, the increased m6A in 3'-UTR mRNA of oncogene CDCP1 was found in malignant transformed cells. Mechanistically, the m6A methyltransferase METTL3 and demethylases ALKBH5 mediate the m6A modification in 3'-UTR of CDCP1 mRNA. METTL3 and m6A reader YTHDF1 preferentially recognize m6A residues on CPCP1 3'-UTR and promote CDCP1 translation. We further showed that METTL3 and CDCP1 are upregulated in the bladder cancer patient samples and the expression of METTL3 and CDCP1 is correlated with the progression status of the bladder cancers. Inhibition of the METTL3-m6A-CDCP1 axis resulted in decreased growth and progression of chemical-transformed cells and bladder cancer cells. Most importantly, METTL3-m6A-CDCP1 axis has synergistic effect with chemical carcinogens in promoting malignant transformation of uroepithelial cells and bladder cancer tumorigenesis in vitro and in vivo. Taken together, our results identify dynamic m6A modification in chemical-induced malignant transformation and provide insight into critical roles of the METTL3-m6A-CDCP1 axis in chemical carcinogenesis.
Project description:Objective:This study aims to uncover the progression of thyroid carcinoma influenced by the m6A methyltransferase METTL3 through regulating m6A methylation on TCF1 mRNA and the activated Wnt pathway. Methods:Thyroid carcinoma tissues and paracancerous ones were collected for detecting levels of METTL3 and TCF1. Potential correlation between levels of METTL3 and TCF1 was analyzed by Pearson analysis. Survival of thyroid carcinoma patients influenced by METTL3 level was assessed by Kaplan-Meier method. Regulatory effect of METTL3 on migratory ability in TPC-1 cells was examined by wound healing assay. The interaction between METTL3 with TCF1 and IGF2BP2 was verified by RNA-Binding Protein Immunoprecipitation (RIP) assay. Meanwhile, the activity of the Wnt pathway was reflected by TOP/FOP-Flash. At last, rescue experiments were conducted to clarify the involvement of TCF1 in phenotype changes of thyroid carcinoma cells that were regulated by METTL3. Results:METTL3 and TCF1 were upregulated in thyroid carcinoma. Similarly, METTL3 was highly expressed in thyroid carcinoma cells as well. Kaplan-Meier method uncovered poor prognosis in thyroid carcinoma patients expressing a high level of METTL3. Silence of METTL3 inhibited migratory ability and Wnt activity in TPC-1 cells. RIP assay confirmed the interaction between TCF1 and METTL3 or IGF2BP2. Moreover, METTL3 positively regulated the enrichment abundance of TCF1 in anti-IGF2BP2. Rescue experiments demonstrated that TCF1 was responsible for METTL3-regulated thyroid carcinoma progression via the m6A methylation. Conclusion:Upregulated m6A methyltransferase METTL3 promotes the progression of thyroid carcinoma through m6A methylation on TCF1.
Project description:Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, whose treatment still has a major challenge of achieving a satisfactory curative effect. The underlying mechanisms also have not been fully illustrated. N 6-Methyladenosine (m6A) has been identified as the most prevalent internal modification of mRNAs present in eukaryotes, which is involved in the pathogenesis of cancers. It remains unclear how m6A mRNA methylation is functionally linked to the pathogenesis of DLBCL. In this study, we sought to explore the roles of METTL3 on DLBCL development. The results showed that m6A level for RNA methylation and the expression level of METTL3 were upregulated in DLBCL tissues and cell lines. Functionally, downregulated METTL3 expression in DLBCL cells inhibited the cell proliferation ability. Further mechanism analysis indicated that METTL3 knockdown abates the m6A methylation and total mRNA level of pigment epithelium-derived factor (PEDF). However, Wnt/?-catenin signaling was not thus activated. Overexpressed PEDF abrogates the inhibition of cell proliferation in DLBCL cells that is caused by METTL3 silence. In summary, the above-mentioned results demonstrated that the METTL3 promotes DLBCL progression by regulating the m6A level of PEDF.
Project description:N6-methyladenosine (m6A) is the most prevalent type of RNA modification. METTL3 in the methyltransferase complex is the core enzyme responsible for methylation. METTL3 selectively catalyzes the adenosines centered in the RRAC motif. Functional studies established that m6A could enhance the translation efficiency (TE) of modified genes by recruiting reader protein YTHDF1 and other initiation factors. We downloaded the m6A peaks in HeLa cells from a previous study and defined the m6A modified genes and sites. Ancestral mutations in the genic region fixed in the HeLa cell samples were defined using their mRNA-Seq data and the alignment between human and mouse genomes. Furthermore, in the small interfering (si)-METTL3 sample, the calculated TE foldchange of all genes was compared to that in the negative control. The TE of m6A genes was globally down-regulated in si-METTL3 versus control compared to the non-m6A genes. In m6A modified genes, RRAC motif mutations were suppressed compared to mutations in non-motif regions or non-m6A genes. Among the m6A genes, a fraction RRAC motif mutations negatively correlated with the TE foldchange (si-METTL3 versus control). The TE of m6A modified genes was enhanced in HeLa cells. RRAC motif mutations could potentially prevent methylation of adenosines and consequently abolish the enhanced translation. Such mutations in the RRAC motif might be deleterious. Accordingly, we observed lower fractions of mutations in RRAC motifs than in other regions. This prevention of mutations in the RRAC motif could be a strategy adopted by cancer cells to maintain the elevated translation of particular genes.
Project description:N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.
Project description:METTL3 catalyzes the formation of N6-methyl-adenosine (m6A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m6A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m6A modification in spermatogenesis and reproduction in mammals.
Project description:The methyltransferase like 3 (METTL3) is a key component of the large N6-adenosine-methyltransferase complex in mammalian responsible for N6-methyladenosine (m6A) modification in diverse RNAs including mRNA, tRNA, rRNA, small nuclear RNA, microRNA precursor and long non-coding RNA. However, the characteristics of METTL3 in activation and post-translational modification (PTM) is seldom understood. Here we find that METTL3 is modified by SUMO1 mainly at lysine residues K177, K211, K212 and K215, which can be reduced by an SUMO1-specific protease SENP1. SUMOylation of METTL3 does not alter its stability, localization and interaction with METTL14 and WTAP, but significantly represses its m6A methytransferase activity resulting in the decrease of m6A levels in mRNAs. Consistently with this, the abundance of m6A in mRNAs is increased with re-expression of the mutant METTL3-4KR compared to that of wild-type METTL3 in human non-small cell lung carcinoma (NSCLC) cell line H1299-shMETTL3, in which endogenous METTL3 was knockdown. The alternation of m6A in mRNAs and subsequently change of gene expression profiles, which are mediated by SUMOylation of METTL3, may directly influence the soft-agar colony formation and xenografted tumor growth of H1299 cells. Our results uncover an important mechanism for SUMOylation of METTL3 regulating its m6A RNA methyltransferase activity.
Project description:N6-methyladenosine (m6A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m6A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m6A, especially gene- and cell-type-specific m6A mRNA modifications. We also show that microRNAs (miRNAs) regulate m6A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m6A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m6A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m6A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m6A formation of mRNAs and provide a foundation for future functional studies of m6A modification in cell reprogramming. m6A-seq in ESC, iPSC, NSC and sertoli cells.
Project description:N6-methyladenosine (m6A) is the richest modification in mammalian messenger RNAs (mRNAs), and exerts key roles in many biological processes, including cancer development, whereas its roles in prostate carcinoma (PCa) remain to be unclear. Here, we found that m6A modifications are increased in PCa and methyltransferase-like 3 (METTL3), but not other major m6A modification genes including METTL14, fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), was the major dysregulated gene associated with abnormal m6A modification. In addition, METTL3 up-regulation acted as a poor prognostic factor for overall survival and disease-free survival in PCa patients. Knockdown of METTL3 significantly inhibited PCa cells proliferation, migration, and invasion. In addition, over-expression of METTL3, but not its catalytic mutant form, significantly promoted PCa cells growth and progression. Mechanistically, we revealed that METTL3 enhanced MYC(c-myc) expression by increasing m6A levels of MYC mRNA transcript, leading to oncogenic functions in PCa. Importantly, PCa cells growth and progression inhibition by METTL3 knockdown were restored through over-expression of MYC. Our results uncovered a METTL3/m6A/MYC axis and provided insight into the mechanisms of PCa progression.
Project description:The dynamic N6-methyladenosine (m6A) modification of mRNA plays a role in regulating gene expression and determining cell fate. However, the functions of m6A mRNA modification in bladder cancer stem cells (BCSCs) have not been described. Here, we show that global RNA m6A abundance and the expression of m6A-forming enzyme METTL3 are higher in BCSCs than those in non-CSCs of bladder cancer (BCa) cells. The depletion of the METTL3 inhibited the self-renewal of BCSCs, as evidenced by decreased ALDH activity and sphere-forming ability. Mechanistically, METTL3 regulates the m6A modification and thereby the expression of AF4/FMR2 family member 4 (AFF4), knockdown of which phenocopies the METTL3 ablation and diminishes the tumor-initiating capability of BCSCs in vivo. AFF4 binds to the promoter regions and sustains the transcription of SOX2 and MYC which have critical biological functions in BCSCs. Collectively, our results demonstrate the critical roles of m6A modification in self-renewal and tumorigenicity of BCSCs through a novel signaling axis of METTL3-AFF4-SOX2/MYC.